Avian leukosis virus (ALV)-induced osteopetrosis is caused by the abnormal growth and differentiation of osteoblasts. To evaluate the role of infection in osteopetrosis induction, the replication of an osteopetrosis-inducing virus (Br21) has been compared in osteopetrotic bone, calvarial-derived osteoblasts, and chick embryo fibroblasts. Much higher levels of infection occurred in diseased bone than in the cultures. Severe cases of osteopetrosis contained 10 times more viral DNA, 30 times more mature capsid protein, 5 to 10 times more Gag precursor protein, and 2 to 3 times more Env protein than the infected cultures. Virus replication in the cultured osteoblasts was similar to that in fibroblasts except for a distinctive asymmetric localization of Gag proteins. In osteopetrotic chickens, bones became atypically enlarged and sera contained elevated levels of osteoblast differentiation markers (alkaline phosphatase and osteocalcin). In cultures, infections did not affect the growth or differentiation of osteoblasts. Thus, the infected cultures lacked aspects of the bone environment that support both the high levels of infection and the aberrant function of osteoblasts characteristic of ALV-induced osteopetrosis.